Difference between revisions of "Part:BBa K3863004"

Revision as of 13:43, 21 October 2021

Phac-flag-PCT(optimized)-HA-his

This is a composite part designed to produce bioplastic PHA with his-tag. PHA synthase 1 from Pseudomonas resinovorans. The protein sequence in this part is based on the K2042001 sequence Evry 2016. PCT is propionate CoA transferase from Clostridum propionicum. The protein sequence in this part is based on the K1211001 sequence Yale 2013. Tackling the issues with biobricks, we have optimized the codon.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1708
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 87
Illegal BamHI site found at 1506
Illegal XhoI site found at 1030
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1060
Illegal NgoMIV site found at 2125
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 251

Team PuiChing_Macau 2021: Bioplastic Production

Validation of construct expression of K3863004

Fig.1a PhaC (BBa_K3863004) qPCR

Fig.1b PCT(optimized) (BBa_K3863004) qPCR

Fig 1. Real-time qPCR fold change of reference gene

To future confirm that our constructs have the expression level, we have performed RT-qPCR The RT-qPCR results show that we have successfully assembled target genes K3863004 to the vector (pETDuet).

Validation of Bioplastic Production

In order to observe the conversion performance, infrared spectroscopic analysis was applied since PHA and PLA have their particular functional group in chemical structure, and are performed in particular wavelengths. The absorption peak of PLA is at 1081, 1188, 1364, 1452 , 1751 cm^-1[1], whereas the absorption peak 979, 1057, 1100, 1282, 1723, 2934, 2977cm^-1[2]. We have compared and analyse the peak of the wavelength to confirm the bioplastic product we have produced. In Figure 2, no absorption peak of PHA and PLA was identified, which indicated that the vector (pETDuet Vector) cannot give the desired product. On the other hand, the absorption peaks of PHA and PLA were identified after incubation, which suggested that the BBa_K3863004 and BBa_K3863008, the bioplastic performs significantly in the transformation process. Furthermore, the absorption peaks of PHA were found, which showed that BBa_K3863007 performs well and was the product that we expected to have. These results have proven that the bioplastic that we have produced can be formed significantly with BBa_K3863004, BBa_K3863008 and BBa_K3863007.

Figure 2a. IR spectrum of pETDuet vector

Figure 2b. IR spectrum of BBa_K3863004

Quantitative Measure of Bioplastic Production

In order to measure the amount of bioplastic produced, we used 20 microgram Nile Red (sigma aldrich N3013) per milliliter of agar to make a Nile agar plate and streak the cell culture (BBa_K3863007(PHA), BBa_K3863004(PCT) , BBa_K3863008(Phasin)) on the plate. After incubating for 2 days at 37 celsius in darkness. Stereomicroscope (Nikon SMZ18) is used to measure the intensity of Nile red.

Fig 3.Mean intensity of Nile red for quantitative measure of bioplastic production of (BBa_K3863007(PHA), BBa_K3863004(PhaC), BBa_K3863008(Phasin))

Reference

[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3450024/pdf/12088_2009_Article_31.pdf
[2]https://www.researchgate.net/figure/Fourier-transform-infrared-FTIR-spectra-of-PLA-PEG-PLA-PEG-blend-and-PLA-PEG-xGnP_fig1_277675367

@@ Line 43: / Line 43: @@
-===Validation of Bioplastic Production===
+===Quantitative Measure of Bioplastic Production===
+<p>In order to measure the amount of bioplastic produced, we used 20 microgram Nile Red (sigma aldrich N3013) per milliliter of agar to make a Nile agar plate and streak the cell culture (BBa_K3863007(PHA), BBa_K3863004(PCT) , BBa_K3863008(Phasin)) on the plate. After incubating for 2 days at 37 celsius in darkness. Stereomicroscope (Nikon SMZ18) is used to measure the intensity of Nile red. </p>
+https://2021.igem.org/wiki/images/c/c5/T--PuiChing_Macau--result0000.jpg
+<p>Fig 3.Mean intensity of Nile red for quantitative measure of bioplastic production of (BBa_K3863007(PHA), BBa_K3863004(PhaC), BBa_K3863008(Phasin))</p>
-<p>In order to observe the conversion performance, infrared spectroscopic analysis was applied since PHA and PLA have their particular functional group in chemical structure, and are performed in particular wavelengths. The absorption peak of PLA is at 1081, 1188, 1364, 1452 , 1751 cm<sup>-1[1]</sup>,  whereas the absorption peak 979, 1057, 1100, 1282, 1723, 2934, 2977cm<sup>-1[2]</sup>. We have compared and analyse the peak of the wavelength to confirm the bioplastic product we have produced.
+==Reference==
-In Figure 2, no absorption peak of PHA and PLA was identified, which indicated that the vector (pETDuet Vector) cannot give the desired product. On the other hand, the absorption peaks of PHA and PLA were identified after incubation, which suggested that the BBa_K3863004 and BBa_K3863008, the bioplastic performs significantly in the transformation process. Furthermore, the absorption peaks of PHA were found, which  showed that BBa_K3863007 performs well and was the product that we expected to have. These results have proven that the bioplastic that we have produced can be formed significantly with BBa_K3863004, BBa_K3863008 and BBa_K3863007.
-</p>
-<h2>Reference</h2>
 <p>[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3450024/pdf/12088_2009_Article_31.pdf<br>
 [2]https://www.researchgate.net/figure/Fourier-transform-infrared-FTIR-spectra-of-PLA-PEG-PLA-PEG-blend-and-PLA-PEG-xGnP_fig1_277675367</p>